Protein and peptide drugs represent a rapidly expanding class of therapeutic molecules (Strohl and Knight, 2009, Curr Opin Biotechnol. 20:668-672). However, the actions of peptidic molecules are limited primarily to extracellular targets (Fischer, 2007, Med Res Rev. 27:755-795; Johnson et al., 2011, Methods Mol Biol. 683:535-551) because the size and composition of most polypeptides and peptide mimetics do not facilitate efficient transport across the plasma membrane into the cytosol or nucleus of mammalian cells (Luedtke et al., 2003, J Am Chem Soc. 125:12374-12375). It has been known for over 40 years that addition of cationic charges to a peptide or protein can aid transport into cells (Ryser and Hancock, 1965, Science. 150:501-503), and many reports have demonstrated the utility of appending basic sequences derived from the HIV Trans-Activator of Transcription (Tat) (Zhou et al., 2009, Cell Stem Cell. 4:381-384), D. melanogaster Antennapedia (Théodore et al., 1995, J. Neurosci. 15:7158-7167), or simply polyarginine (for example, Arg8; SEQ ID NO:68) (Futaki et al., 2001, J Biol Chem. 276:5836-5840) to peptides or small molecules (Wender et al., 2008, Adv Drug Deliv Rev 60:452-472) to increase ‘cell uptake’. Certain highly positively charged proteins (Cronican et al., 2011, Chem Biol. 18:833-838) and toxins (Johannes and Popoff, 2008, Cell. 135:1175-1187) are also ‘taken up’ by cells with varying levels of efficiency.
Two contrasting mechanisms have been proposed for the cytosolic entry of cationic proteins and related molecules. The first (ion pair-guided passive diffusion) posits that guanidinium side chains on the polypeptide form hydrogen bonds with cell surface phospholipids creating neutral ion pairs that passively diffuse across the plasma membrane (Rothbard et al., 2005, Adv Drug Deliv Rev. 57:495-504). The second model (endosomal release), asserts that endocytosis is a major portal through which cationic polypeptides and peptide mimetics enter the cell (Fischer, 2007, Med Res Rev. 27:755-795). Previous investigations have attempted to distinguish between these two models by blocking endocytosis, via thermal (Derossi et al., 1996, J Biochem. 271:18188-18193), pharmacologic (Wadia et al., 2004, Nat Med. 10:310-315; Fischer et al., 2004, J Biol Chem. 279:12625-12635), or genetic means (Ter-Avetisyan et al., 2008, J Biol Chem 284:3370-3378). The interpretation of these experiments is complicated, however, by differences in protein/polypeptide concentration and analytical method. For example, incubation of living cells with cationic proteins/polypeptides at concentrations ≥10 μM leads to the formation of nucleation zones (Duchardt et al., 2007, Traffic. 8:848-866) that transiently disrupt membranes (Palm-Apergi et al., 2009, FASEB J 23:214-223), causing the spontaneous release of peptide into the cytosol. Incubation of cells at lower concentrations (≤5 μM) of peptide, in the presence of drugs that inhibit endocytosis, prevents cytoplasmic access (Wadia et al., 2004, Nat Med. 10:310-315), implying that at low concentrations, the molecules studied cannot diffuse through the plasma membrane. Moreover, the many studies using microscopy to examine cells fixed by treatment with formaldehyde or methanol must be reevaluated in light of evidence that the fixation process can release fluorescently labeled peptides from endosomes (Belitsky et al., 2002, Bioorg Med Chem 10:3313-3318; Richard et al., 2003, J Biol Chem. 278:585-590), an artifact not observed during microscopic examination of living cells. Finally, the high-intensity light used during microscopy can itself facilitate the redistribution of fluorescently labeled peptides from endosomes to cytoplasm (Maiolo et al., 2004, J Am Chem Soc. 126:15376-15377). Unfortunately, most cationic peptides and proteins that engage the endocytic machinery remain trapped within vesicles where they are topologically separated from the cell interior and unable to access targets in the cytosol or nucleus (Erazo-Oliveras et al., 2012, Pharmaceuticals 5:1177-1209). Intracellular function, when observed, is believed to result from the mechanistically indistinct, unpredictable, and inefficient process of endosomal escape. Thus, whether, when, and how these cationic molecules escape endocytic vesicles to access the cytosol remain unanswered questions.
Attempts to identify structural determinants of cell permeability are complicated by the above experimental details as well as the fact that neither Tat (SEQ ID NO:67) nor Arg8 (SEQ ID NO:68) possesses a defined fold. Miniature proteins are a family of small (36-aa), well-folded polypeptides that adopt a characteristic hairpin fold consisting of axially packed α- and PPII helices (Blundell et al., 1981, Proc Natl Acad Sci USA. 78:4175-4179; Hodges and Schepartz, 2007, J Am Chem Soc. 129:11024-11025). Other small well-folded polypeptides include those with a zinc finger fold and others known as talens. Miniature proteins identified through both rational design (Zondlo et al., 1999, J Am Chem Soc. 121:6938-6939; Zellefrow et al., 2006, J Am Chem Soc. 128:16506-16507) and molecular evolution (Chin and Schepartz, 2001, Chem. Int. Ed. Engl. 40:3806-3809; Rutledge et al., 2003, J Am Chem Soc. 125:14336-14347; Golemi-Kotra et al., 2004, J Am Chem Soc. 126:4-5; Gemperli et al., 2005, J Am Chem Soc. 127:1596-1597) can modulate protein function by inhibiting protein interactions (Rutledge et al., 2003, J Am Chem Soc. 125:14336-14347; Gemperli et al., 2005, J Am Chem Soc. 127:1596-1597); both loss of function and gain of function activities have been observed (Golemi-Kotra et al., 2004, J Am Chem Soc. 126:4-5; Gemperli et al., 2005, J Am Chem Soc. 127:1596-1597; Zellefrow et al., 2006, J Am Chem Soc. 128:16506-16507). It has been reported previously that minimally cationic miniature proteins containing between 2 and 6 arginine residues embedded within the α- or PHI helix were taken up by mammalian cells in culture more efficiently than Tat (SEQ ID NO:67) or Arg8 (SEQ ID NO:68) (Daniels and Schepartz, 2007, J Am Chem Soc. 129:14578-14579; Smith et al., 2008, J Am Chem Soc. 130:2948-2949).
Despite advances made in the art, there remains a need in the art for improved peptides, proteins and fusion molecules capable of efficiently crossing biological membranes with low toxicity. The present invention fulfills this need.